U.S. patent number 6,294,361 [Application Number 08/958,768] was granted by the patent office on 2001-09-25 for processes for photoreactive inactivation of a virus in blood cell or coagulation factor containing compositions and use thereof for preparing compositions useful for transfusion.
This patent grant is currently assigned to New York Blood Center, Inc.. Invention is credited to Nicholas E. Geacintov, Bernard Horowitz, Henrietta Nunno, Shanti B. Rywkin, Jay E. Valinsky, Bolanle Williams.
United States Patent |
6,294,361 |
Horowitz , et al. |
September 25, 2001 |
Processes for photoreactive inactivation of a virus in blood cell
or coagulation factor containing compositions and use thereof for
preparing compositions useful for transfusion
Abstract
The present invention concerns a process for inactivating an
extracellular lipid enveloped human pathogenic virus and/or an
intracellular human pathogenic virus which may be present in a
blood cell composition containing .gtoreq.1.times.10.sup.9 cells/ml
by contacting that composition with a virucidally effective amount
of at least one photoreactive compound having an absorption maximum
of .gtoreq.630 nm, light and oxygen and/or a quencher. In one
embodiment of the invention, the process is conducted under
conditions whereby a structural integrity of greater than 80% of at
least one type of blood cell contained within said composition is
retained. Another embodiment of the invention relates to a process
for inactivating an extracellular lipid enveloped human pathogenic
virus and/or an intracellular human pathogenic virus which may be
present in a composition containing at least one coagulation factor
while retaining at least 77% of said coagulation factor by
contacting said composition with a virucidally effective amount of
at least one photoreactive compound, light and a quencher. The
processes of the invention can be used to prepare blood products,
which, in turn, are suitable for transfusion into a recipient in
need of such transfusion.
Inventors: |
Horowitz; Bernard (New
Rochelle, NY), Valinsky; Jay E. (New York, NY),
Geacintov; Nicholas E. (New York, NY), Williams; Bolanle
(New York, NY), Rywkin; Shanti B. (Brooklyn, NY), Nunno;
Henrietta (New York, NY) |
Assignee: |
New York Blood Center, Inc.
(New York, NY)
|
Family
ID: |
27061415 |
Appl.
No.: |
08/958,768 |
Filed: |
October 26, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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031787 |
Mar 15, 1993 |
6077659 |
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706919 |
May 29, 1991 |
5232844 |
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524208 |
May 15, 1990 |
5120649 |
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Current U.S.
Class: |
435/173.3;
435/173.1; 435/2; 435/236 |
Current CPC
Class: |
A61K
41/17 (20200101); A61K 41/0023 (20130101); A61K
41/0038 (20130101); C12N 7/00 (20130101); A61L
2/0011 (20130101); C12N 2760/20263 (20130101); C12N
2740/16063 (20130101) |
Current International
Class: |
A61L
2/00 (20060101); A61K 41/00 (20060101); C12N
7/04 (20060101); C12N 7/06 (20060101); C12N
013/00 (); C12N 007/04 (); A01N 001/02 () |
Field of
Search: |
;435/173.1,173.3,236,2
;424/90 |
References Cited
[Referenced By]
U.S. Patent Documents
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4693981 |
September 1987 |
Wiesehahn et al. |
|
Other References
Lin et al. (1989), Blood, 74(1), 517-525.* .
Swartz et al. (1979) Proc. Soc. Exp. Biol. Med., 161, 204-209.*
.
Bodylak et al. (1983) J. Clin. Microbiol., 17(2), 374-376.* .
Sonada et al. (1987) Photochem. Photobiol., 46(5), 625-631.* .
Rosenthal et al. (1989) in "Pholocyanines," Lezhoff et al. (EDS),
pp. 397-425, VCH Publishers, Inc., New York..
|
Primary Examiner: Weber; Jon P.
Attorney, Agent or Firm: Amster, Rothstein &
Ebenstein
Government Interests
GOVERNMENT RIGHTS
This invention was made with United States government support under
Grant 1-RO1-HL41221 from the NHLBI. The United States Government
has certain rights in this invention.
Parent Case Text
This is a continuation of application Ser. No. 08/031,787 filed on
Mar. 15, 1993, now U.S. Pat. No. 6,077,659.
Which is a division, of application Ser. No. 07/706,919, filed May
29, 1991, now U.S. Pat. No. 5,232,844, which is a CIP of U.S. Ser.
No. 07/524,208 filed May 15, 1990, now U.S. Pat. No. 5,120,649.
Claims
What is claimed is:
1. A process for inactivating an extracellular lipid enveloped
human pathogenic virus and/or an intracellular human pathogenic
virus which may be present in a blood cell composition, comprising
contacting said composition with a virucidally effective amount of
a photoreactive compound having an absorption maximum of
.gtoreq.630 nm, light and a quencher, wherein said composition
contains .gtoreq.2.25.times.10.sup.9 cells/ml.
2. A process for inactivating an extracellular lipid enveloped
human pathogenic virus and/or a intracellular human pathogenic
virus which may be present in a blood cell composition containing
.gtoreq.2.25.times.10.sup.9 cells/ml while retaining structural
integrity of greater than 80% of one type of blood cell contained
within said composition, comprising contacting said composition
with a virucidally effective amount of at least one photoreactive
compound having an absorption maximum of .gtoreq.630 nm, light and
a quencher.
3. The process according to claim 2, wherein the blood cell
composition comprises at least one component selected from the
group consisting of red blood cells, platelets and whole blood.
4. The process according to claim 3, wherein the red blood cells
and/or platelets are concentrated.
5. The process according to claim 2, wherein the type of blood cell
is red blood cells and the structural integrity of said red blood
cells is ascertained by assessing the amount of hemoglobin released
after treatment of said composition with said photoreactive
compound, light and a quencher, a release of less than 20% of
hemoglobin indicates that the structural integrity of greater than
80% of said red blood cells was retained after treatment with said
photoreactive compound, light and a quencher.
6. The process according to claim 2, wherein the type of blood cell
is platelets and the structural integrity of said platelets is
ascertained by counting the number of platelets remaining after
treatment of said composition with said photoreactive compound,
light and a quencher, a retention of greater than 80% of said
platelets indicates that the structural integrity of greater than
80% of said platelets was retained after treatment with said
photoreactive compound, light and a quencher.
7. The process according to claim 2, wherein oxygen is present
during the process.
8. The process according to claim 2, wherein the light is visible
light at a wave length of 630 nm-700 nm.
9. The process according to claim 2, wherein the photoreactive
compound is phthalocyanine.
10. The process according to claim 2, wherein the extracellular
lipid enveloped human pathogenic virus and/or the intracellular
human pathogenic virus is a human immunodeficiency virus (HIV).
11. A process for preparing a blood cell composition suitable for
transfusion into a recipient in need of such transfusion which
comprises inactivating an extracellular lipid enveloped human
pathogenic virus and/or an intracellular human pathogenic virus
which may be present in said composition according to the process
of claim 2.
12. A process for inactivating a human immunodeficiency virus (HIV)
which may be present in a blood cell composition containing
.gtoreq.2.25.times.10.sup.9 cells/ml while retaining structural
integrity of greater than 80% of one type of blood cell contained
within said composition, comprising contacting said composition
with a virucidally effective amount of a photoreactive compound
having an absorption maximum of .gtoreq.630 nm, light and a
quencher.
13. A process for inactivating an extracellular lipid enveloped
human pathogenic virus and/or an intracellular human pathogenic
virus which may be present in a composition containing a
coagulation factor while retaining .gtoreq.94% of said coagulation
factor, consisting essentially of contacting said composition which
a virucidally effective amount of a photoreactive compound, light
and a quencher.
14. The process according to claim 13, wherein the photoreactive
compound is a psoralen and the light is UVA.
15. The process according to claim 13, wherein the quencher is
glutathione.
16. The process according to claim 13, wherein the photoreactive
compound is phthalocyanine and the light is visible light at a
wavelength of 630 nm to 700 nm.
17. The process according to claim 13, wherein said composition is
human plasma.
18. The process according to claim 13, wherein the coagulation
factor is Factor VIII.
19. The process according to claim 13, wherein the extracellular
lipid enveloped human pathogenic virus and/or the intracellular
human pathogenic virus is a human immunodeficiency virus (HIV).
20. A process for preparing a blood cell composition suitable for
transfusion into a recipient in need of such transfusion which
comprises inactivating an extracellular lipid enveloped human
pathogenic virus and/or an intracellular human pathogenic virus
which may be present in said composition according to the process
of claim 13.
21. A process for inactivating a human immunodeficiency virus (HIV)
which may be present in a composition containing at least one
coagulation factor while retaining .gtoreq.94% of said coagulation
factor, comprising contacting said composition with a virucidally
effective amount of at least one photoreactive compound having an
absorption maximum of .gtoreq.630 nm, light and a quencher.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns a method to inactivate viruses in
biological compositions, for example, in whole blood or red blood
cell concentrates, without incurring substantial disruption or
inactivation of cells, for example, without adversely affecting red
blood cell structure or function, by using a photoactive compound,
for example, a phthalocyanine, together with light exposure and
variations thereon.
2. Description of Related Art
Nature of the Concept
Substantial progress has been made in reducing the viral
infectivity of whole blood and its components through improved
donor selection and donor blood screening procedures. Despite this
progress, there is a continued risk of transmission of viruses
including hepatitis viruses and human immunodeficiency viruses
(HIV) by whole blood and blood products.
The risk of transmission of certain viruses (e.g., hapatitis B
virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus
(HIV)) has been considerably reduced and possibly eliminated in
coagulation factor concentrates through the application of
virucidal procedures during the course of manufacture (Prince, A.
M., Horowitz, B., Horowitz, M. S., Zang, E., "The Development of
Virus-Free Labile Blood Derivatives--A Review", Eur. J. Epidemiol.,
1987; 3:103-118 and Mannucci, P. M., Colombo, M., "Virucidal
Treatment of Clotting Factor Concentrates", The Lancet,
1988;782-785). However, when treating coagulation factor
concentrates, some viruses (e.g., parvovirus) may remain
infectious. In addition, the development of virucidal processes
applicable to cell components, i.e., blood cell fractions such as
red blood cells or platelets, has been slow, both because cells are
more fragile than proteins, and cells serve to harbor and protect
virus against inactivation. Nonetheless, if virus transmission by
whole blood or blood components is to be eliminated, effective
virus removal or potent virucidal methods applicable to blood cells
will be required. Since both red blood cells or platelets are
non-replicating, approaches directed toward nucleic acid
modification might offer the required specificity.
It is important to recognize in assessing virucidal procedures for
cell-containing solutions that virus will be present in multiple
forms: virus free of the cell; formed virus associated with the
cell; functional, but unpackaged viral nucleic acid within the
cell; and viral nucleic acid integrated into the cell genome. Each
form should be considered infectious and capable of causing viral
disease in vivo. Virucidal methods which inactivate virus in one
form, e.g., cell-free virus, may not inactivate virus in other
forms, e.g., cell-associated forms. Additionally, the presence of
cells is known to inhibit the action of both physical and chemical
approaches to virus inactivation. Cells compete for added virucidal
reagents and absorb radiation which otherwise would be virucidal.
Thus, for example, while ultraviolet irradiation is highly
virucidal in salt solutions or in dilute protein solution, the
degree of virucidal activity is incomplete when treating
cell-containing solutions. Furthermore, in this context, it is not
sufficient to inactivate virus alone; rather, it is necessary to do
so with sufficient vigor that viral infectivity is eliminated
without deleterious effects to the valuable cell components, e.g.,
red blood cells.
Most virucidal procedures which have been developed, e.g.,
pasteurization or solvent/detergent treatment, cannot be applied to
blood cell preparations without damaging the cells and rendering
them unfit for transfusion.
Heretofore, it has not been possible to prepare virus sterilized
forms of whole blood or red cell concentrates or platelet
concentrates where at least 10.sup.4 infectious units (ID.sub.50)
and preferably .gtoreq.10.sup.6 ID.sub.50 of both intracellular and
extracellular virus were inactivated without adversely affecting
the cells and/or without using highly toxic agents.
Phthalocyanines
While there has been growing interest in the use of phthalocyanines
for the treatment of cancerous cells (Rosenthal, I. and Ben Hur,
E., "Phthalocyanines in Photobiology", Lezhoff C. C. and Lever A.
B. P. eds., Phthalocyanines, VCH Publishers, Inc., New York, N.Y.,
1989, 393-425), phthalocyanines are generally thought of as being
hemolytic, making applicants' results herein all the more
surprising. For example, Ben-Hur and Rosenthal ("Photohemolysis of
Human Erthrocytes Induced by Aluminum Phthalocyanine
Tetrasulfonate", Cancer Lett., 30: 321-327, 1986) studied the
photohemolysis of human red blood cells induced by aluminum
phthalocyanine tetrasulfonate. Substantial (20-100%) hemolysis was
induced by treatment with 2.5 to 10 .mu.M AlPCS.sub.4 at all light
fluences.gtoreq.40 KJ/m.sup.2 (.gtoreq.4 J/cm.sup.2). Ben-Hur and
Rosenthal did not address the problem of virus kill. Similarly,
Sonoda, Krishna and Riesz ("The Role of Singlet Oxygen in the
Photohemolysis of Red Blood Cells Sensitized by Phthalocyanine",
Photochem. Photobiol., 46: 625-631, 1987) studied the
photohemolysis induced by each of several phthalocyanine
derivatives. Aluminum and zinc phthalocyanines were each hemolytic,
while free (no metal) phthalocyanine or those with iron, copper or
cobalt as the central metal cation were not. Virus kill was not
studied.
Singer et al (C. R. J. Singer, T. Azim and Q. Sattentau,
"Preliminary Evaluation of Phthalocyanine Photosensitization For
Inactivation Of Viral Pathogens in Blood Products", [abstract]
British J. Hematology, Mar. 23-25, 1988:Abs. 31), in what is
believed to be the only study on virus kill performed with
phthalocyanines, demonstrated that an unstated quantity of both
Epstein Barr virus and of HIV added to plasma was inactivated on
treatment with 5 and 25 .mu.g/mL of sulfonated aluminum
phthalocyanine and 2 mW/cm.sup.2 for 30 minutes (3.6 J/cm.sup.2).
Factor VIII recovery was only 50%. Singer et al reported no actual
work on cells or cell-associated virus, though they state that
application to red cells is being evaluated. Given the relatively
poor recovery (50%) of factor VIII reported by Singer, the greater
fragility of cells than proteins, and the previous experience on
the photohemolysis of red cells to treatment with phthalocyanine,
the results herein are all the more surprising.
Other Lipophilic Dyes in the Treatment of Whole Blood or Red Blood
Cell Concentrates
Cole et al (Cole, M., Stromberg, R., Friedman, L., Benade, L.,
Shumaker, J., "Photochemical Inactivation of Virus in Red Cells",
Transfusion, 29, Supp:42s Abs., 1989) explored the use of
merocyanine 540 in the treatment of packed red blood cells diluted
to a hematocrit of 15%. When plasma was removed such that its
concentration was 2.6%, a 6 log reduction of vesicular stomatitis
virus was achieved. However, only a 1 log reduction in VSV was
achieved in samples containing 15% plasma. The authors concluded
that "although plasma is required to protect the red blood cells
from damage, viral kill is also significantly reduced". This
conclusion is supported by the observation that the presence of 5%
albumin inhibited virus kill in suspension of washed platelets
(Prodouz, K. N., "Effect of Merocyanine 540 on Platelet Function
and Reduction of its Antiviral Activity by Albumin", Transfusion,
29, Supp:42s Abs., 1989), and that though 6 logs of model viruses
in buffer could be inactivated by merocyanine 540, only 1-3 logs of
virus could be inactivated in the presence of 12-25% plasma
(Moroff, G., Benade, L. E., Dabay, M., George, V. M., Shumaker, J.
and Dodd, R. Y., "Use of Photochemical Procedures to Inactivate
Viruses in Platelet Suspensions", Transfusion, 29, Supp:S15 Abs.,
1989). Furthermore, the authors stated that the procedure
"adversely affected platelet properties".
Matthews, J. T., Newman, J. T., Sogandares-Bernal, F., et al,
"Photodynamic Therapy of Viral Contaminants with Potential For
Blood Banking Applications", Transfusion, 1988;28:81-83 studied
treatment of whole blood with hematoporphyrin derivatives and
light. They reported the inactivation of 3.times.10.sup.5 PFU of
herpes simplex virus type 1 (HSV-1) on treatment of culture medium
with 2.5 .mu.g/mL dihematoporphyrin ether (DHE) and light, but only
the inactivation of 10.sup.3 PFU on treatment of blood under the
same condition. Increasing the concentration of DHE to 20 .mu.g/ml
did not improve virus kill. While red blood cell structure and
function was well maintained at 2.5 .mu.g/mL DHE and light at 5
J/cm.sup.2, cell-free HIV (2.times.10.sup.3 ID.sub.50) added to
buffer alone was not completely killed under this condition.
Other Photoactive Compounds
Lin et al (Lin, L., Wiesehahn, G. P., Morel, P. A. and Corash, L.,
"Use of 8-Methoxypsoralen and Long-wavelength Ultraviolet Radiation
for Decontamination of Platelet Concentrates", Blood, 74:517-525,
1989) demonstrated that psoralen and exposure to UV-A inactivated
.gtoreq.10.sup.5.5 ID of feline leukemia virus added to a platelet
concentrate; however, studies in whole blood or red cell
concentrates were not performed. Platelet morphology, aggregation,
and the release reaction were well maintained immediately following
treatment, and showed comparable values when compared to untreated
controls on storage for up to 96 hours. In contrast, Moroff et al
(Moroff G., Benade, L. E., Dabay, M., George, V. M., Shumaker, J.
and Dodd, R. Y., "Use of Photochemical Procedures to Inactivate
Viruses in Platelet Suspensions", Transfusion, 29, Supp, S15 Abs.,
1989) explored the use of psoralen for the treatment of platelets
and concluded that the presence of as little as 12% plasma
inhibited virus kill and that platelet properties were adversely
affected. It should be pointed out that, as typically prepared, red
blood cells and platelet concentrates for transfusion are suspended
in 100% plasma.
Other Agents
In U.S. patent application Ser. No. 07/279,179, filed Dec. 2, 1988,
and in a recent published abstract (Williams et al, Blood, 1988,
72: Suppl.:287a), vesicular stomatitis virus added to whole blood
was reported to be inactivated on incubation with a hydrolyzable,
aryl diol epoxide without causing red cell lysis.
Ozone has been asserted to decontaminate whole blood containing
10.sup.9 pFU/mL of hepatitis virus (Zee, Y. C. and Bolton, D. C.,
"Ozone Decontamination of Blood and Blood Products", U.S. Pat. No.
4,632,980). However, no data were provided in support of this
allegation.
Prodouz, K. N., Fratantoni, J. C., Boone, J. E. and Bonner R. F.,
"Use of Laser-UV for Inactivation of Virus in Blood Products",
Blood, 1987; 70:589-592 reported that laser-UV treatment of a
platelet concentrate largely maintained platelet function under
conditions where up to 10.sup.6 (ID.sub.50) of polio virus was
inactivated. However, virus inactivation was studied in buffered
medium alone and not in the presence of platelets, and only a
cell-free form of the virus was employed.
Hartman et al (Hartman, F. W., Mangun, G. H., Feeley, N., Jackson,
E., "On the Chemical Sterilization of Blood and Blood Products",
Proc. Soc. Exp. Biol. Med., 70:248-254, 1949) showed that treatment
of whole blood with the nitrogen mustard,
methyl-bis(beta-chloroethyl) amine hydrochloride resulted in the
inactivation of 10.sup.6.6 ID.sub.50 of vesicular stomatitis virus
under conditions where red cell hemolysis was not greater than the
control. It should be pointed out that nitrogen mustards are
carcinogens.
LoGrippo (LoGrippo, G. A., "Investigations of the Use of
Beta-Propiolactone in Virus Inactivation", Ann. NY Aca. Sci., 83,
578-594, (1959)) treated red cells separately from plasma with
beta-propiolactone. Treatment resulted in the inactivation of more
than 10.sup.8 ID.sub.50 of Eastern equine encephalitis virus
without causing red cell hemolysis. Subsequent injection of the
treated red cells in man resulted in a shortened circulatory
half-life.
SUMMARY OF THE INVENTION
It is an object of the present invention to inactivate viruses in
cell-containing compositions without incurring substantial
disruption or inactivation of cells.
It is another object of the present invention to inactivate viruses
in whole blood, red blood cell concentrates and platelet
concentrates, without adversely affecting red cell or platelet
structure or function.
It is another object of the present invention to inactivate viruses
in biological compositions without incurring substantial
inactivation of desired, soluble biological substances (e.g.,
coagulation factor concentrates, hemoglobin solutions).
It is a further object of this invention to improve virus safety in
blood banks of both whole blood, red blood cell concentrates and
platelet concentrates, and any products derived from whole blood,
red blood cell concentrates or platelet concentrates.
It is still a further object of the invention to reduce exposure to
hospital care workers and other health care workers to viruses to
which they otherwise would be exposed.
It is still a further object of the invention to reduce the
circulating viral burden in animals and man.
It is still a further object of the invention to improve the
storage properties of cell-containing compositions, e.g., red cell
concentrate, prior to use.
The above objects, as well as other objects, aims and advantages
are satisfied by the present invention.
The present invention concerns a method of inactivating excellular
lipid enveloped human pathogenic viruses or intracellular human
pathogenic viruses in a cell-containing composition without
incurring substantial disruption or inactivation of cells,
comprising contacting a cell-containing composition having
.gtoreq.1.times.10.sup.9 cells/ml and containing infectious virus
with a virucidally effective amount of at least one photoreactive
compound having an absorption maximum of .gtoreq.630 nm, light and
an oxidizer to substantially inactivate the virus and to result in
a retention of intact cell functionality and structure of greater
than 80%.
In accordance with another aspect of the invention, extracellular
and intracellular virus in a biological composition is inactivated
without incurring substantial disruption or inactivation of the
composition, by a process comprising contacting said composition
with a virucidally effective amount of at least one photoreactive
compound, light, and a quencher thereby to inactivate said virus
while retaining functionality of said substance.
More particularly, the present invention concerns a method for
inactivating extracellular as well as intracellular viruses in
whole blood, red blood cell concentrates, platelet concentrates or
products derived from whole blood or red blood cell concentrates or
platelet concentrates comprising contacting said whole blood, red
blood cell concentrates or products derived from whole blood or red
blood cell concentrates with an effective virucidal amount of a
photoreactive compound having an absorption maximum greater than
630 nm, for example, a purpurin or a phthalocyanine, and exposing
the resultant composition to light in the presence of an oxidizer,
together with the optional presence of a quencher, for example,
glutathione.
The present invention also concerns a composition comprising human
red blood cells suitable for transfusion at concentration of
.gtoreq.1 to 10.sup.9 cells/ml and having all extracellular lipid
enveloped and intracellular human pathogenic viruses in a
non-infectious form, the red cells preferably having a normal
recovery on infusion of 70% or greater, preferably 85% or greater
and having a satisfactory circulatory survival, e.g., for red blood
cells of .gtoreq.20 days and preferably for 30 days.
The aforesaid compositions preferably have a greater resistance to
osmotic shock than normal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 comprises four graphs depicting the inactivation of
cell-free vesicular stomatitis virus (VSV) with aluminum
phthalocyanine chloride (AlPc). FIG. 1a depicts the results using
whole blood. FIG. 1b depicts the results using a red blood cell
concentrate. FIG. 1c depicts the results using whole blood diluted
5-fold with PBS. FIG. 1d depicts the results using a red blood cell
concentrate diluted 2-fold with PBS.
FIG. 2 comprises four graphs depicting the inactivation of
cell-free VSV with sulfonated aluminum phthalocyanine. FIG. 2a
depicts the results using AlPcS.sub.2 with whole blood. FIG. 2b
depicts the results using AlPcS.sub.2 with a red blood cell
concentrate. FIG. 2c depicts the results using AlPcS.sub.4 with
whole blood. FIG. 2d depicts the results using AlPcS.sub.4 with a
red blood cell concentrate.
FIG. 3 is a graph depicting erythrocyte osmotic fragility prior to
and following treatment with AlPc.
DETAILED DESCRIPTION OF THE INVENTION
Blood is made up of solids (cells, i.e., erythrocytes, leucocytes,
and platelets) and liquid (plasma). The cells are transfused in the
treatment of anemia, clotting disorders, infections, etc. In
addition, the cells contain potentially valuable substances such as
hemoglobin, and they can be induced to make other potentially
valuable substances such as interferon, growth factors, and other
biological response modifiers. The plasma is composed mainly of
water, salts, lipids and proteins. The proteins are divided into
groups called fibrinogens, serum globulins and serum albumins.
Typical antibodies (immune globulins) found in human blood plasma
include those directed against infectious hepatitis, influenza H,
etc.
Blood transfusions are used to treat anemia resulting from disease
or hemorrhage, shock resulting from loss of plasma proteins or loss
of circulating volume, diseases where an adequate level of plasma
protein is not maintained, for example, hemophilia, and to bestow
passive immunization.
With certain diseases one or several of the components of blood may
be lacking. Thus the administration of the proper fraction will
suffice, and the other components will not be "wasted" on the
patient; the other fractions can be used for another patient. The
separation of blood into components and their subsequent
fractionation allows the cells and/or proteins to be concentrated,
thus enhancing their therapeutic use.
Cell types found in human blood include red blood cells, platelets
and several types of leukocytes. Methods for the preparation of
cell concentrates useful in transfusion can be found in Kirk
Othmer's Encyclopedia of Chemical Technology, Third Edition,
Interscience Publishers, Volume 4, pp 25-37, the entire contents of
which are incorporated by reference herein.
Proteins found in the blood cell fraction include hemoglobin,
fibronectin, fibrinogen, platelet derived growth factor, superoxide
dismutase, enzymes of carbohydrate and protein metabolism, etc. In
addition, the synthesis of other proteins can be induced, such as
interferons and growth factors.
A comprehensive list of inducible leukocyte proteins can be found
in Stanley Cohen, Edgar Pick, J. J. Oppenheim, "Biology of the
Lymphokines", Academic Press, New York, (1979).
The present invention is directed to contacting at least one
photoreactive compound with a cell-containing composition such as
whole blood, red blood cell concentrates, platelet concentrates,
platelet extracts, leukocyte concentrates, semen, ascites fluid,
milk, lymphatic fluid, hybridoma cell lines and products derived
from any of the above.
The present invention can be employed to treat the product of a
composition containing a non-blood normal or cancerous cells or the
product of gene splicing.
When quenchers are not added, suitable photosensitizer compounds
for use in the present invention include phthalocyanines, purpurins
and other molecules which resemble the porphyrins in structure,
although some of the atoms in the basic porphyrin-like frame (as
well as their arrangement) may vary. For example, phthalocyanines
are porphyrin-like (azaporphyrins), except that the tetrapyrrole
ring linked by methine carbon atoms in porphyrins is replaced by
four isoindole units linked by aza nitrogen atoms. These
phthalocyanine, porphyrin, and purpurin molecules may or may not
contain metallo or metalloid central atoms, and various
substitutents may be placed on the basic molecular framework to (a)
red-shift the longest wavelength absorption maxima beyond 630 nm,
(b) increase the molar extinction coefficient to enhance the
absorptivity of the exciting red light, and (c) modulate the
solubilities of the photosensitizer molecules in aqueous
environments, as well as their lipophilicities, or DNA-binding
abilities.
Photoreactive compounds for use in the present invention which
contain metals, for example, germanium or gallium, are diamagnetic,
rather than paramagnetic.
Photosensitizers, including substituted photosensitizers, which can
be utilized in the present invention will result in compounds
having the following characteristics:
(a) a molar extinction coefficient of .gtoreq.50,000 Molar.sup.-1
cm.sup.-1 ;
(b) an absorption maximum of .gtoreq.630 nm, preferably 660 to
730;
(c) a solubility of .gtoreq.1 .mu.M in both water and apolar
solvents;
(d) having amphiphilic characteristics;
(e) soluble in aqueous saline buffer solutions at the
concentrations of use in a time frame of approximately 2 hours.
Preferred photoreactive compounds for use in the present invention
are phthalocyanines (Pc's or PC's). Phthalocyanines are
porphyrin-like compounds which are chemically stable, well defined,
and easily synthesized (Spikes, J., "Phthalocyanines as
Photosensitizers in Biological Systems and for the Photodynamic
Therapy of Tumors", Photochemistry and Photobioloqy,
1986;43:691-699 and Ben-Hur, E. and Rosenthal, I., "The
Phthalocyanines: A New Class of Mammalian Cells Photosensitizers
with a Potential for Cancer Phototherapy", Int. J. Radiat. Biol.,
1989;47:145-147). There is encouraging evidence in the literature
indicating the lack of toxicity of phthalocyanine dyes to mammals
(Moser, F. H. and Thomas, A. C., The Phthalocyanines, Boca
Raton:CRC Press, 1984). The phthalocyanines have very strong
electronic absorption bands at wavelengths above 630 nm. Hemoglobin
has a relatively low absorbance in this spectral region.
Non-limiting examples of phthalocyanines for use in the present
invention include the following:
zinc tetrasulfophthalocyanine,
tetrasulfophthalocyanine,
aluminum tetranitrophthalocyanine,
zinc-tetranitrophthalocycyanine,
tetracarboxyphthalocyanine,
GaCl-tetrasulfophthalocyanine,
AlCl-tetrasulfophthalocyanine,
Ga-tetrasulfophthalocyanine and
GaCl-, AlCl- or Ga-tetranitrophthalocyanine.
In a preferred embodiment of the invention, aluminum
phthalocyanines are employed. Preferred aluminum phthalocyanines
include aluminum phthalocyanine chloride (AlPc) and sulfonated
forms of aluminum phthalocyanine, e.g., AlPcS.sub.2 and
AlPcS.sub.4. Zinc can replace aluminum as the central atom, and the
ring can be nitrated instead of sulfonated.
When quenchers are added, suitable photosensitizer compounds for
use in the present invention include phthalocyanines, purpurins,
and other molecules which resemble the porphyrins in structure (as
described above) as well as photoactive compounds excited by
ultraviolet light (e.g., psoralen, 8-methoxypsoralen,
4'-aminomethyl-4,5',8-trimethyl psoralen, bergapten, and
angelicin), and dyes which absorb light in the visible spectrum
(e.g., hypericin methylene blue, eosin fluoresceins and
flavins).
Suitable quenchers are any substances known to react with free
radicals or reactive forms of oxygen, more specifically which
decrease the efficiencies of photodynamically catalyzed chemical
reactions (e.g. DNA strand breads), or decrease the cytoxicity in
photodynamic cell killing experiments. In accordance with the
present invention, however, surprisingly quenching is effected
without substantial decrease in virucidal action.
Representative quenchers include unsaturated fatty acids, reduced
sugars, cholesterol, indole derivatives, and the like, azides, such
as sodium azide, tryptophan, polyhydric alcohols such as glycerol
and mannitol, thiols such as glutathione, superoxide dismutase,
quercetin, DABCO, and the like. The use of mixtures of quenchers is
also contemplated.
The quencher is used in conventional quenching amounts but
surprisingly, when used, the overall process results in
preferential damage to the virus but not the desired biological
material.
Non-limiting examples of lipid coated, human pathogenic viruses
that can be inactivated by the present invention include vesicular
stomatitis virus (VSV), Moloney sarcoma virus, Sindbis virus, human
immunodeficiency viruses (HIV-1; HIV-2), human T-cell
lymphotorophic virus-I (HTLV-I), hepatitis B virus, non-A, non-B
hepatitis virus (NANB) (hepatitis C), cytomegalovirus, Epstein Barr
viruses, lactate dehydrogenase elevating virus, herpes group
viruses, rhabdoviruses, leukoviruses, myxoviruses, alphaviruses,
Arboviruses (group B), paramyxoviruses, arenaviruses and
coronaviruses. Non-limiting examples of non-enveloped viruses that
can be inactivated by the present invention include parvirus, polio
virus, hepatitis A virus and enteric non-A, non-B hepatitis
virus.
The process of the present invention is preferably conducted at 0
to 45.degree. C., and most preferably at 4 to 37.degree. C. for up
to 48 hours and preferably for 2 to 24 hours.
The process of the invention is preferably conducted in a neutral
pH range of 6.3 to 7.7. A typical light fluence range for the
invention is 5 to 500 J/cm.sup.2, preferably 100 to 500 J/cm.sup.2
with phthalocyanine and 5 to 100 J/cm.sup.2 with psoralen. The
brighter the light, the less time is required. With flowing
systems, very bright light for short times would be employed. For
blood bags, longer times and less bright light can be used.
Preferably the concentration of the photoreactive compound in the
absence of quenchers is 1 to 100 .mu.M; for red cell concentrates,
the concentration of the photoreactive compound is most preferably
10 to 25 .mu.M. When quenchers are used, the concentrations of
photoactive compounds are those typically employed, e.g. 25
.mu.g/ml for AMT.
The process of the present invention is carried out in the presence
of an oxidizer. Oxygen is a nonlimiting example of an oxidizer for
use in the present invention. The concentration of oxygen can be
the endogenous quantity, or can be modified by placement of the
material being treated in an atmosphere designed to lower or raise
oxygen concentration.
Cell-containing compositions to be treated according to the
invention have .gtoreq.1.times.10.sup.9 cells/ml, preferably
.gtoreq.5.times.10.sup.9 cells/ml and most preferably
.gtoreq.1.times.10.sup.10 cells/ml. Furthermore, cell-containing
compositions to be treated according to the invention have
preferably .gtoreq.4 mg/ml protein and more preferably .gtoreq.25
mg/ml protein and most preferably 50 to 60 mg/ml protein (unwashed
cells).
Non-cell containing compositions to be treated according to the
invention have .gtoreq.0.1 mg/ml and preferably .gtoreq.5 mg/ml
protein.
In the inventive process, at least 10.sup.4, preferably 10.sup.6,
infectious units of virus are inactivated.
The inventive process results in improved storage stability, i.e.,
treated cells that can be stored in liquid or frozen form and for
which reduced cell destruction is obtained.
The cell-containing composition according to the invention, while
initially containing .gtoreq.1000 infectious units of virus/L,
after the virus has been inactivated and treatment according to the
invention has been conducted, has a retention of intact cell
functionality and structure of greater than 80%, preferably greater
than 90% and most preferably greater than 98%.
By the inactivation procedure of the invention, most if not
virtually all of the viruses contained therein would be
inactivated. A method for determining infectivity levels by
inoculation into chimpanzees (in vivo) is discussed by Prince, A.
M., Stephen, W., Bortman, B. and van den Ende, M. C., "Evaluation
of the Effect of Beta-propiolactone/Ultraviolet Irradiation
(BPL/UV) Treatment of Source Plasma on Hepatitis Transmission by
Factor IX Complex in Chimpanzees", Thrombosis and Hemostasis, 44:
138-142, (1980).
According to the invention, inactivation of virus is obtained to
the extent of at least "4 logs", preferably .gtoreq.6 logs, i.e.,
virus in the sample is totally inactivated to the extent determined
by infectivity studies where that virus is present in the untreated
sample in such a concentration that even after dilution to 10.sup.4
(or 10.sup.6) viral activity can be measured.
The present invention describes inactivating viruses, while
simultaneously retaining labile blood cell functional and
structural features.
Functional activities of red cells are ascertained by measurements
of metabolite levels, enzymatic activities, electrolyte levels and
oxygen carrying capacity. Structural integrity of red cells is
assessed by measurements of hemoglobin release, osmotic fragility,
survival in vivo following radiolabeling with chromium-51,
antigenicity and by evaluation of modification of cell surface
proteins.
Functional activities of platelets are determined by their ability
to aggregate in the presence of certain biological agents and their
morphology. Structural integrity of platelets is assessed by in
vivo survival following radiolabeling with indium-11 and
identification of the presence of specific platelet antigens.
The method of the present invention can be used in conjunction with
other viral inactivating agents, e.g., beta-propiolactone or UV or
other forms of radiation, e.g., gamma rays.
The present invention demonstrates the following:
(1) photoreactive compounds such as phthalocyanines together with
light exposure can inactivate viruses in whole blood or red cell
concentrates, without adversely affecting red cell structure or
function,
(2) a lipophilic dye with an absorption maximum of .gtoreq.630 nm
can inactivate large quantities (e.g., .gtoreq.10.sup.5.5
ID.sub.50) of virus in whole blood or a red cell concentrate under
conditions which maintain red cell structure and function,
(3) both extracellular and intracellular virus present in whole
blood, a red cell concentrate or a platelet concentrate can be
inactivated without adversely affecting cell structure or function,
and
(4) a lipophilic dye on exposure to light can stabilize red blood
cells to osmotic injury.
(5) the inclusion of a quencher of a photochemically catalyzed
reaction during or following said reaction reduces cell or protein
damage which may occur without substantially reducing virus
kill.
The principal advantage of the phthalocyanines over other
lipophilic dyes such as hematoporphyrin derivative is the extremely
strong optical absorption of phthalocyanines at 630-700 nm. Light
at this wavelength has improved tissue penetrating properties, as
compared with the shorter wavelength of light absorbed by the usual
porphyrin and hematoporphyrin sensitizers. Furthermore, the
absorption spectrum of phthalocyanines is better separated from
that of blood components, especially hemoglobin, which has an
absorption maximum at 578 nm.
As reported herein, photocatalyzed reactions with hydrophobic dyes
results in inactivation of extracellular enveloped viruses such as
VSV and HIV, while extracellular encephalomyocarditis virus (EMC),
a non-enveloped virus, was not inactivated. In addition,
AlPcS.sub.2 and AlPcS.sub.4, which bind to the more hydrophilic
regions of the cell, were more effective virucidal agents than AlPc
at a similar concentration. It is important to note that both
cell-free and cell-associated viruses were inactivated under the
conditions examined, and that red cell integrity was maintained, as
judged by the absence of hemoglobin release (<2%) on treatment,
or following storage. In fact, treatment of a red cell suspension
with AlPc and light stabilized the red cell against hypotonic
shock. Further evidence of the integrity of AlPcS.sub.4 -treated
red blood cells comes from the measurement of their circulatory
half-life. Treated baboon red blood cells had a half-life of 13.4
days while untreated baboon red blood cells had a half-life of 13.9
days.
That VSV added to an entire red blood cell concentrate unit was
inactivated indicates that a procedure based on AlPc addition and
exposure to light can be implemented in a blood banking
environment. Treatment of collected units in a light cabinet,
perhaps for a period as long as 6 to 24 hours, or for briefer
periods if multiple or more intense light sources are employed, is
envisioned.
In a preferred embodiment of the present invention, a light fluence
of 250 to 1000 J/cm.sup.2 is applied to a sample 2 to 4 cm thick
and agitation is utilized. In a further preferred embodiment of the
invention, the process according to the invention is applied to a
sample in a blood bag.
After treatment with the photoreactive compound, excess
photoreactive compound can be removed by centrifugation, washing
and/or dialysis.
In an embodiment of the present invention, the treated
cell-containing fraction from the inventive process is transfused
or returned to the donor, e.g., human donor, from which the initial
cell-containing fraction was derived. In this manner, the level of
circulating virus in the donor will be reduced, thus improving the
donor's ability to clear virus and/or improving the efficacy of
antiviral drugs.
As noted hereinabove, the invention also extends to an inactivation
method involving a photoactive compound, light and a quencher, with
or without an oxidizer.
The inclusion of a quencher during AMT/UVA treatment of a platelet
concentrate in the presence of oxygen resulted in normal platelet
function, as measured in standard aggregation reaction, and the
inactivation of .gtoreq.10.sup.5.5 TCID.sub.50 of VSV; without
quencher addition, the rate and extent of platelet aggregation was
reduced. Surprisingly, virus kill was similar in both samples.
Similarly, the inclusion of a quencher during AMT/UVA treatment of
blood plasma resulted in the quantitative recovery of coagulation
factor VIII; without quencher addition, the recovery was only 77%.
Again, surprisingly, there was no difference in VSV kill. In yet
another example, the circulatory half-life of rabbit red blood
cells was assessed following treatment with aluminum phthalocyanine
tetrasulfonate and visible light. Without adversely affecting virus
kill, inclusion of a quencher resulted in prolongation of the
circulatory survival of treated red blood cells.
The invention will now be described with reference to the following
non-limiting examples.
EXAMPLES
Materials and Methods
Blood
Whole blood was typically less than 48 hours old when used. Prior
to use, it was stored at 4.degree. C. Red blood cell concentrates
(RBCC) were prepared from whole blood by centrifugation for 20
minutes at 2000 r.p.m. with removal of most of the plasma layer.
Where indicated, whole blood was diluted 5-fold or the red blood
cell concentrates were diluted 2-fold with phosphate buffered
saline (PBS; Gibco Laboratories, Grand Island, N.Y.).
Aluminum Phthalocyanine Solutions
Aluminum phthalocyanine chloride (AlPc) was purchased from Kodak
Laboratory Chemicals, Rochester, N.Y. Stock solutions of AlPc
(0.01M) were prepared in spectrophotometric grade
N,N-dimethylformamide (Aldrich, Milwaukee, Wis.). Aluminum
phthalocyanine tetrasulfonate (AlPcS.sub.4) and aluminum
phthalocyanine disulfonate (AlPcS.sub.2) were purchased from
Porphyrin Products Inc., Logan, Utah. Stock solutions of
AlPcS.sub.2 and AlPcS.sub.4 (6.2.times.10.sup.-4 M) were prepared
in PBS. The concentration of all phthalocyanine solutions was
determined spectro-photometrically using a molar extinction
coefficient of 2.times.10.sup.5 1 mol.sup.-1 cm.sup.-1 at the
absorption maximum at 670 nm for AlPc, 674 nm for AlPcS.sub.2 and
675 nm for AlPcS.sub.4.
Psoralen Solutions
4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was purchased from
HRI Assoc. Inc., Concord, Calif. Stock solutions of AMT (4 mg/ml)
were prepared in distilled water.
Model Virus Studies
The inactivation of the following viruses was studied: vesicular
stomatitis virus (VSV), a lipid enveloped, RNA virus;
encephalomyocarditis virus (EMC), a protein enveloped, RNA virus;
and human immunodeficiency virus (HIV), a human, pathogenic
retrovirus.
VSV was cultured in human A549 cells. EMC stocks were prepared in
mouse L929 or human A459 cells. Culturing and assay procedures were
similar to those described in Horowitz, B., Wiebe, M. E., Lippin,
A. and Stryker, M. H., "Inactivation of Viruses in Labile Blood
Derivatives", Transfusion, 1985;25:516-522. Infectivity of VSV and
EMC was assessed by endpoint, 10-fold serial dilutions in DMEM
culture medium (Gibco Laboratories, Grand Island, N.Y.) with 10%
fetal calf serum (FCS; MA Bioproducts, Walkersville, Md.). Each
dilution was used to inoculate eight replicate wells of human A549
cells in 96-well microtiter plates. Virus-induced cytopathology was
scored after 72 hours of incubation at 37.degree. C. in 5%
CO.sub.2. The reported virus titer was calculated using the
Spearman-Karber method (Spearman, C., "The Method of Right and
Wrong Cases' (`Constant Stimuli`) Without Gauss's Formula", Br. J.
Psychol., 1908;2:227-242) and indicates the quantity of virus which
infect s 50% of the tissue culture wells (TCID.sub.50).
Cell -associated VSV was prepared by incubating a confluent
monolayer of humanA549 cells with 5 ml of 10.sup.7 ID.sub.50 /ml
VSV in serum-free DMEM for 1 hour at 37.degree. C. under 5%
CO.sub.2 in 150 cm.sup.2 tissue culture flasks. The multiplicity of
infection under these conditions was approximately 2.1 TCID.sub.50
/cell. After decanting off the liquid, the attached cells were
washed three times to remove free virus with 50 ml PBS per wash.
Afterwards, 40 ml of DMEM containing 5% FCS were added, and the
cells were incubated for an additional 4 3/4 hours. The attached
cells were washed three times with PBS and released by treatment
for 10 minutes with a normal saline solution containing 0.01%
trypsin (Cooper Biomedical, Freehold, N.J.; two times crystallized)
and 5 .mu.g/ml EDTA. The released cells were collected by
centrifugation, washed three times with PBS and resuspended in
PBS.
To assess inactivation, cell-free virus was added to the blood
component being studied at a 1:10 dilution, and 3 ml aliquots of
this mixture were distributed in polystyrene tubes (Fisher
Scientific, Springfield, N.J.; Cat. #2027; 7 ml capacity) followed
by the addition of the phthalocyanine derivative. The samples were
mixed continuously using a hematology mixer (Fisher Scientific,
Cat. #14-060-1) and photoirradiated with light from a Solar
Simulator (Oriel Corp., Stratford, Conn.) fitted with a Zenith 300
watt Xe short arc lamp equipped with an amber, 570 nm long-pass
filter (Oriel Corp.). The light power at the sample was about 25-26
mWatts/cm.sup.2 as measured with a photometer (Model No. IL1350
International Light, Newburyport, Mass.) with a detector (Model No.
SED038) fitted with a wide band pass filter (F#8174) and a diffuser
(W#4425). As compared with the data presented below, the filtration
through a 676 nm interference filter (the Optometrics Corp.,
Catalog No. 02-6765, Ayer, Mass.) placed on the detector permitted
the transmission of 1.3% of the light power. Irradiation times were
typically 30, 60 and 120 minutes corresponding to fluences of 44,
88 and 176 J/cm.sup.2 respectively. A constant flow of air was
provided by a fan, and the temperature of the sample did not rise
above 28.degree. C. during irradiation.
Virus inactivation of an entire red blood cell concentrate (RBCC)
unit was carried out in a 600 ml capacity #5J359 bag (Fenwall
Division, Deerfield, Ill.). A Thermolyne Speci-mix mixer model
M26125 (Sybron Corp., Iowa) was used to mix the sample in the bag
during photoirradiation.
For assessment of virus inactivation, the reaction was stopped by
10-fold dilution into DMEM containing 5% fetal calf serum, and the
red blood cells were removed by centrifugation at 1500 rpm for 10
minutes. The lack of virus inactivation at this dilution or in the
absence of light was confirmed for each of the inactivation
conditions studied. Samples were sterile filtered (Swinnex filters,
Millipore Corp., Bedford, Mass.) and frozen at -70.degree. C. or
below until assay.
The procedures for the assessment of the inactivation of
cell-associated VSV were similar to those of cell-free VSV, except
all experiments with cell-associated VSV were carried out under
totally controlled aseptic conditions. At the conclusion of the
experiment, the infected A549 cells were isolated with the addition
of Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, N.J.) and
centrifuged in a swinging bucket rotor at 1800 xg for 30 minutes at
ambient temperature. The layer containing the A549 cells was
collected, washed three times with PBS by centrifugation,
resuspended in 1 ml PBS and immediately assayed for VSV infectivity
by endpoint, 10-fold serial dilutions as with cell-free virus.
Assessment of HIV Inactivation
The HTLV III.sub.b strain of human immunodeficiency virus (HIV) was
used in these experiments. Measurement of infectivity was similar
to that reported previously (Prince, A. M., Pascual, D., Kosolapov,
L. B., et al, "Prevalence, Clinical Significance, and Strain
Specificity of Neutralizing Antibody to the Human Immunodeficiency
Virus", The Journal of Infectious Diseases, 1987;156:268-272). Ten
thousand-fold concentrates of cell-free HIV, prepared by continuous
flow sucrose banding, were purchased from Bionetics, Inc.
(Rockville, Md.). Titrations were carried out with serial, ten-fold
dilutions in microtiter plates using RPMI 1640 containing 10% FCS,
with either CEM or H9 cells at a concentration of 8.times.10.sup.5
/mL. Before use, cells were conditioned by incubation for 1 hour at
37.degree. C. in the above medium containing 2 .mu.g/mL of
polybrene. Virus in treated samples was adsorbed to cells for 2
hours at 37.degree. C. in the dark. Cultures were then washed twice
in medium by centrifuging plates for 10 minutes at 2000 rpm and
aspirating the supernatants in order to remove the treating
compound and reduce toxicity. 150 .mu.l cultures were then fed with
25 .mu.l of medium at 4, 7 and 10 days. At 14 days, cultures were
washed twice with PBS (phosphate buffered saline) to remove viral
antigens carried over from the inoculum, and the cells were lysed
in PBS containing 0.5% Triton X-100. Lysates were assayed for HIV
p55 antigens by ELISA using plates coated with rabbit antiserum
against recombinant p55 (Syntex Corp., Palo Alto, Calif.) and
peroxidase labeled rabbit anti-p55. This assay had essentially the
same sensitivity in measurement of p24 as the Dupont p24 antigen
assay.
To increase the sensitivity for measurement of small quantities of
residual virus, 0.5-1.0 ml of undiluted virus-containing fluids
treated with AlPc were also inoculated into 5 ml macro-cultures,
and were fed by removing half volumes and replacing with fresh
medium twice weekly for 4 weeks.
For cell-associated HIV, 25 ml culture of 8.times.10.sup.5 /mL CEM
of H9 cells were inoculated with 10.sup.4 TCID.sub.50 of HIV.
Cultures were fed by removal of half volumes and replacement with
fresh medium twice weekly. At each feeding, supernatant fluids were
assayed for p55 antigens by ELISA. When the titer reached 1:64 or
greater, usually at 10-12 days, the infected cells were used in the
following experiments. Prior to use in experiments, aliquots of
10.sup.6 infected cells were pelleted and resuspended in 100 .mu.L
of HIV immune globulin (Prince, A. M., Horowitz, B., Baker, L. et
al, "Failure of an HIV Immune Globulin To Protect Chimpanzees
Against Experimental Challenge With HIV", PNAS, 1988;85:6944-6948),
incubated for 1 hour at 37.degree. C., and washed three times in
culture medium in order to reduce the amount of non-cell associated
virus. Infected cells were then suspended in medium, or whole blood
anticoagulated with CPD (citrate phosphate dextrose), to a
concentration of 10.sup.6 /mL. These mixtures were exposed to
varying concentrations of AlPc with or without exposure to light.
After treatment, samples were diluted 1:2 with RPMI-1640 and
centrifuged through Ficoll-Hypaque to separate lymphocytes from
erythrocytes. The recovered lymphocytes were washed three times,
counted, and serially diluted in 100 .mu.l of medium. Uninfected
CEM cells were then added and the cultures processed as for the
infectivity titration described above.
Red Cell Measurements
Total hemoglobin was quantitated using Drabkin's reagent (Sigma
Procedure No. 525, Sigma Diagnostics, St. Louis, Mo.). Plasma
hemoglobin was assessed, after removal of cells by centrifugation,
by measuring the optical density of the plasma at A540 and assuming
an absorbance of 0.86 for a 1 mg/ml solution (Antonini, E. and
Brunori, M., "Hemoglobin and Myoglobin in Their Reactions with
Ligands", Amsterdam: North-Holland Publishing Co., 1971. (Neuberger
A., Tatum E. L., eds., Frontiers of Biology; Vol. 21)). Prior to
centrifugation, red cell concentrates were diluted 1:1 with PBS.
The results were expressed as a percentage of the total hemoglobin
present. Osmotic fragility of treated red blood cells was measured
as previously described in Dacie, J. V., Lord, M. B., Vaughan, J.
M. and Oxon, D. M., "The Fragility of Red Blood Cells, Its
Measurements and Significance", J. Path Bact., 1938, 46:341-356. pH
measurements were made with a PHM 82 pH meter (Radiometer America
Inc., Cleveland, Ohio). The circulatory half-life of autologous
rabbit red blood cells was determined by washing the treated red
blood cells to remove plasma proteins and labeling the cells with
.sup.51 Cr.
Example 1
Inactivation of VSV and EMC By AlPc
The inactivation of cell-free VSV added to whole blood
(5.times.10.sup.9 red blood cells/ml) or a red cell concentrate
(1.times.10.sup.10 red blood cells/ml) in the presence of AlPc was
dependent on its concentration and the fluence (dosage) of light
(FIG. 1). Cell-free VSV and AlPc at the indicated concentration
were added to whole blood (FIG. 1a), a red blood cell concentrate
(FIG. 1b), whole blood diluted 5-fold with PBS (FIG. 1c) and a red
blood cell concentrate diluted 2-fold with PBS (FIG. 1d). The
plasma protein concentration in whole blood and the red cell
concentrate was 60 mg/mL prior to the dilution indicated. Samples
(3 mL) were exposed to a constant intensity of light (25-26
mWatts/cm.sup.2) for a varying duration such that the total light
fluence was 44 J/cm.sup.2 (closed circles), 88 J/cm.sup.2 (open
circles), or 176 J/cm.sup.2 (open triangles). Following exposure to
light, virus infectivity was assessed as described herein.
Complete inactivation of VSV (.gtoreq.10.sup.4.0 to 10.sup.4.5
TCID.sub.50) added to whole blood was observed at an AlPc
concentration of 10 AM and a light fluence of 88 and 176
J/cm.sup.2, corresponding to a light intensity of 25
mwatts/cm.sup.2 and exposure times of 60 and 120 minutes,
respectively. At a fluence of 44 J/cm.sup.2, complete inactivation
of added VSV required an AlPc concentration of 25 .mu.M (FIG. 1a).
The inactivation of VSV added to a red blood cell concentrate (RBC
concentration=1.times.10.sup.10 /ml; FIG. 1b) was similar to that
observed in whole blood (FIG. 1a). Complete inactivation of VSV
added to whole blood first diluted 5-fold (FIG. 1c) or to a red
blood cell concentrate first diluted 2-fold (FIG. 1d) with PBS
occurred at a lower AlPc concentration for a given light fluence
than that observed with their undiluted counterparts. VSV
inactivation did not occur in the absence of AlPc or in the dark
(data not shown).
Red blood cell integrity, as determined by hemoglobin released
during the treatment period, was well maintained (lysis >2%)
under each of the conditions presented in FIG. 1.
Cell-free EMC, a non-enveloped virus, was not inactivated on
treatment with AlPc when evaluated under conditions similar to
those described above (data not shown).
Example 2
Intracellular VSV was prepared as described hereinabove. Comparison
of the concentration of cells harvested following trypsin treatment
(2.07.times.10.sup.7 /ml) to viral infectious units
(1.times.10.sup.6 TCID.sub.50 /50 .mu.l; 2.0.times.10.sup.6
TCID.sub.50 /ml) suggests that virtually every cell contained
infectious virus. This intracellular VSV, added to a red blood cell
concentrate, was completely inactivated (.gtoreq.10.sup.5.6
TCID.sub.50) on treatment of this red cell concentrate with 10
.mu.M AlPc and 88 J/cm.sup.2 (Table I). Comparison with the results
reported with cell-free virus (FIG. 1b) indicates that inactivation
of the cell-associated form is more difficult. Red blood cell
structure and function were unaffected.
TABLE I Inactivation of Intracellular VSV Added to a Red Cell
Concentrate with AlPc AlPc Concentration VSV Titer(log.sub.10)
(.mu.M) Dark Light* Log.sub.10 Kill 0 5.2 5.1 0.0 0 5.0 3.6 1.5 2
5.1 .ltoreq.-0.5 .gtoreq.5.6 5 5.3 .ltoreq.-0.5 .gtoreq.5.6 10 Av
5.1 *88 J/cm.sup.2
Example 3
The inactivation of cell-free VSV in the presence of the di- and
tetra-sulfonated derivatives of AlPc was also examined. Cell-free
VSV and AlPcS.sub.2 (FIG. 2a and FIG. 2b) or AlPcS.sub.4 (FIG. 2c
and FIG. 2d) at the indicated concentration were added to whole
blood (FIG. 2a and FIG. 2c) or a red blood cell concentrate (FIG.
2b and FIG. 2d). Other details are as described above with respect
to FIG. 1. Complete inactivation (.gtoreq.10.sup.4 TCID.sub.50) of
VSV with the sulfonated derivatives occurred at a lower AlPc
concentration for a given light fluence than that observed with the
non-sulfonated form (FIG. 1 vs. FIG. 2). Complete inactivation of
VSV added to either whole blood diluted 5-fold or a red cell
concentrate diluted 2-fold with PBS was observed with 2 .mu.M of
either sulfonated derivative and a light fluence of 44 J/cm.sup.2
(data not shown). With regard to hemoglobin release during the
course treatment, little (.ltoreq.2%) was observed at AlPcS.sub.x
concentrations up to 25 .mu.M and a light fluence up to 176
J/cm.sup.2 (Table II).
TABLE II Percent Hemoglobin Released on Treatment of A Red Cell
Concentrate with AlPc Derivatives AlPc Light Fluence Percent
Hemoglobin Released Derivative (J/cm.sup.2) AlPc conc: 5 .mu.m 10
.mu.M 25 .mu.M AlPc 88 1.1 0.8 1.1 176 1.5 0.9 1.4 AlPcS.sub.2 88
0.6 0.5 0.3 176 0.6 0.5 0.4 AlPcS.sub.4 88 0.2 0.5 0.3 176 0.3 0.5
0.3
Example 4
Inactivation of HIV By AlPc
HIV in either a cell-free or intracellular form was added to either
whole blood or a red cell concentrate in a test tube. Treatment of
cell-free HIV used 1.0 .mu.M AlPc and 176 J/cm.sup.2 ; treatment of
intracellular HIV used 5 .mu.M AlPc and 44 J/cm.sup.2. At the
conclusion of treatment, the samples were processed as described
above and HIV antigen measurements were made.
Treatment of whole blood or a red cell concentrate with AlPc was
shown to inactivate .gtoreq.10.sup.4.2 TCID50 of cell-free and
.gtoreq.10.sup.3.6 TCID.sub.50 of a intracellular HIV (Table III).
Red blood cell structure and functions were unaffected.
TABLE III INACTIVATION OF HIV Log.sub.10 Inactivation Cell-Free
Intracellular Whole Blood .gtoreq.4.2 .gtoreq.3.6 Red Cell
Concentrate .gtoreq.4.2 not done
Example 5
Red Blood Cell Integrity
Typical results of the percent hemoglobin released from red blood
cells during the course of treatment with AlPc derivatives are
given in Table II. The percent released varied between 0.2 and 1.5%
of the total hemoglobin present.
The erythrocyte osmotic fragility following treatment of whole
blood was measured with no prior removal of AlPc (FIG. 3). Whole
blood was treated with 10 .mu.M AlPcCl and a light fluence of 44
J/cm.sup.2 (open circles), 88 J/cm.sup.2 (open triangles) and 176
J/cm.sup.2 (open squares). Following treatment and with no
subsequent processing, the erythrocyte osmotic fragility was
determined in these samples and in the untreated control (closed
circles) by dilution into solutions of saline at the indicated
concentration. Following incubation for 30 minutes and
centrifugation, released hemoglobin was measured with Drabkin's
solution and compared with that released on dilution into distilled
water.
As compared with the untreated control, treatment with 10 .mu.M
AlPc at light fluences of 44, 88 and 176 J/cm .sup.2 increased the
resistance of the red cells to osmotic shock.
Example 6
To evaluate the storage stability of treated red cells, 3 ml of
whole blood were treated with 10 .mu.M AlPc and a light fluence of
176 J/cm.sup.2, as in Example 1. Following a storage period of 17
days, released hemoglobin was 1.8% of the total and the pH of the
sample was 6.9, indicative of excellent storage compatibility.
Example 7
A study of VSV inactivation in an intact red cell concentrate unit
was conducted. A red cell concentrate contained in a Fenwal 5J359
blood bag was illuminated from one side only. The inactivation of
all detectable cell-free VSV (.gtoreq.10.sup.4.5 TCID.sub.50) was
achieved with 10.5 .mu.M AlPc and a light fluence of 264 J/cm.sup.2
corresponding to a treatment duration of 3 hours (Table IV). A
10-fold more sensitive macroculture assay did not show the presence
of VSV (kill .gtoreq.10.sup.5.5 TCID.sub.50) at 352 and 396
J/cm.sup.2. Less than 2% lysis was observed even at 396
J/cm.sup.2.
TABLE IV Inactivation of Cell-Free VSV Added to An Individual Red
Cell Concentrate Unit AlPc Concentration Light Fluence VSV Titer
(.mu.M) (J/cm.sup.2) (log.sub.10 TCID.sub.50) 0 0 4.0 10.5 88 2.8
10.5 176 1.3 10.5 264 .ltoreq.-0.5 10.5 352 .ltoreq.-0.5 10.5 396
.ltoreq.-0.5
Example 8
An assessment of the effect of phthalocyanine treatment on platelet
function was conducted. Zinc phthalocyanine (ZnPc) in
dimethylformamide was added to a platelet concentrate containing
5.36.times.10.sup.9 platelets/mL and a plasma protein concentration
of 60 mg/mL. The final concentration of ZnPc was 20 .mu.M. At the
indicated times, platelet count was determined, platelet morphology
was assessed by measurement of mean volumes, and the ability of
platelets to aggregate on addition of adenosine diphosphate (ADP)
was assessed (Table V). Through the total treatment time, platelet
count was maintained to the extent of 86-91%. The mean volume of
the platelets was unaffected. Aggregation in response to ADP,
expressed either in terms of the initial rate of aggregation or the
extent of aggregation was unchanged as compared with the DMF only
control for 10 minute light exposure, though somewhat decreased for
15 and 30 minute light exposures.
TABLE V Effect of Zinc Phthalocyanine on Platelets Aggregation
Platelet Response Count Mean Platelet Initial Maximum
(.times.10.sup.-9 / Volume Rate Extent Sample mL) (micron.sup.3)
(A/min) (A) Controls No light, no ZnPc 5.36 6.1 38 45 Solvent (DMF)
only 5.65 6.4 34 26 Test ZnPC + 10 minute light 4.74 6.3 35 28 ZnPC
+ 15 minute light 4.87 6.1 25 22 ZnPC + 30 minute light 4.63 5.8 25
20
Example 9
The Effect of Red Cell Concentration on AlPcS.sub.4 -Induced
Lysis
Human red blood cells were washed twice with phosphate buffered
saline to remove plasma and then diluted to the indicated
concentration. Aluminum phthalocyanine tetrasulfonate (10 .mu.M)
was added to each and the samples irradiated as described above
with 88 J/cm.sup.2. Following irradiation, the degree of lysis was
determined by the amount of hemoglobin released. As shown in Table
VI, 100% lysis was observed with a red cell concentration of
4.5.times.10.sup.8 cells/ml, but improved suddenly when the cell
concentration was raised to 2.25.times.10.sup.9 cells/ml or
higher.
TABLE VI Percent Hemolysis As a Function of Red Cell Concentration
Red Cell Conc Percent (cells/ml) Hemolysis 9 .times. 10.sup.9 2.9
4.5 .times. 10.sup.9 2.9 2.25 .times. 10.sup.9 2.7 4.5 .times.
10.sup.8 100
Example 10
Comparison Of Virus Kill Of Aluminum Phthalocyanine With
Hematoporphyrin Derivative
Vesicular stomatitis virus (VSV) was added to whole blood followed
by either hematoporphyrin derivative (HPD), a dye with an
absorption maximum below 630 nm, or aluminum phthalocyanine
sulfonate. Each was exposed to light as above. The results (Table
VII below) indicate that virus kill is both faster and more
complete with AlPcS.sub.4 than with HPD.
TABLE VII Conc VSV Kill (log.sub.10) Compound (.mu.M) 30 min 120
min HPD* 18 1.3 1.4 36 1.4 2.4 54 1.7 4.0 AlPcS.sub.4 5 .gtoreq.4.0
.gtoreq.4.0 *Assumed MW of 1106
Example 11
Circulatory half-life of autologous, rabbit red blood cells treated
with aluminum phthalocyanine derivatives in the presence of
quenching agents.
A rabbit red blood cell concentrate (RBCC) containing
1.times.10.sup.10 RBC/ml suspended in plasma was mixed with
aluminum phthalocyanine tetrasulfonate (AlPcS.sub.4) and, where
indicated, a quenching agent added. The mixture was then exposed to
25 mW/cm.sup.2 of visible light for 30 minutes, after which the
treated RBCC was washed by centrifugation, labeled with .sup.51 Cr,
and administered intravenously into the rabbit of origin. The
results indicate that RBC treated in the presence of the added
quenching agent had near normal circulatory in vivo survival
whereas the RBC treated without the added quenching agent had a
shortened in vivo circulatory survival (Table VIII).
The aluminum phthalocyanine derivative and quenching agent were
added to a separate RBCC sample containing vesicular stomatitis
virus and exposed to similar irradiation conditions. The
inactivation of virus was unaffected by the addition of quencher
(Table VIII).
TABLE VIII In vivo circulatory survival of rabbit red blood cells
after treatment with AlPcS4 Half-life Log.sub.10 VSV Dye Quencher
(days) Kill 10 .mu.M AlPcS4 4 mM glutathione 12.5 0 (Dark Control)
10 .mu.M AlPcS4 NONE 3.75 .gtoreq.5.5 10 .mu.M AlPcS4 1 mM mannitol
6 .gtoreq.5.5 10 .mu.M AlPcS4 1 mM tryptophan 8 .gtoreq.5.5 10
.mu.M AlPcS4 1 mM glutathione 10.5 .gtoreq.5.5
Example 12
Aggregation response of platelets treated with a psoralen
derivative in the presence of absence of added quenching agent.
The virucidal and functional effects on platelet concentrates were
assessed after the addition of 25 .mu.g/ml of
4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with and without the
presence of the quenching agent, reduced glutathione (GSH). The
samples were irradiated with 6.5 mW/cm.sup.2 of UVA light for 20-30
min. in the presence of oxygen. Platelet aggregation extent and
rate in response to collagen, measured 24 hours after treatment,
improved in the presence of GSH (Table IX). This was especially
evident with 30 min. UVA exposure, conditions needed to achieve
complete virus kill.
TABLE IX The effect of the addition of GSH on the extent/rate of
aggregation in platelets after UVA treatment with AMT. Aggregation
Exposure Platelet (% control) Log.sub.10 VSV AMT .mu.g/ml GSH Time
Extent Rate Kill 25 0 20 min 98 75 4.1 25 1 mM 20 min 100 84 4.5 25
0 30 min 88 63 .gtoreq.5.5 25 1 mM 30 min 98 90 .gtoreq.5.1
Example 13
Improved process recovery of coagulation factors on treatment of
plasma with AMT and UVA with addition of quenchers.
Human plasma was treated with 25 .mu.g/ml of AMT and exposed to 6.5
mW/cm.sup.2 of UVA light for 20 minutes. Virus kill measurement and
process recovery of coagulation factors were compared after
treatment with and without the addition of glutathione. In the
absence of reduced glutathione (GSH), or in the presence of 1 mM
and 4 mM GSH, the extent of virus kill was 5.1, 5.3 and 5.3
log.sub.10, respectively. While AHF recovery in the absence of GSH
was only 77%, recovery increased to essentially 100% in the
presence of 4 mM GSH.
TABLE X Process recovery of coagulation factor VIII following
treatment of human plasma with AMT and quenching agent. GSH
Concentration NONE 1 mM 4 mM Virus Kill (TCID.sub.50) 5.1 5.3 5.3
Factor VIII Recovery Untreated (u/ml) 1.24 -- -- Treated (u/ml)
0.95 1.16 1.26 % Recovery 77% 94% 102%
Example 14
The enhancement in the recovery of plasma coagulation factors
treated to inactivate viruses with AMT; reduction of oxygen content
versus use of quenchers.
Human plasma was treated with 25 .mu.g/ml AMT under normal
atmospheric conditions in the presence and absence of 1 mM
glutathione added as a quencher, versus treatment under reduced
oxygen concentration through exchange with nitrogen gas. Normally
aerated samples were irradiated with UVA light for 20 minutes while
deoxygenated samples had to be irradiated for 120 minutes to
provide approximately the same virus kill. The data indicate that,
as compared to treatment of normally aerated plasma in the absence
of quencher, (1) the addition of 1 mM glutathione enhances AHF
recovery without compromising virus kill, (2) a high level of virus
kill and a high recovery of coagulation factor recovery can be
achieved by reducing oxygen content, provided the treatment period
is extended, and (3) the use of quencher obviates the need to
perform a gas exchange and reduces the duration of treatment
required for a high level of virus kill.
TABLE XI The recovery of coagulation factor VIII in plasma on
treatment with AMT; effect of quencher addition versus
deoxygenation. UVA VSV AHF AMT Duration Kill Recovery Treatment
Conditions (.mu.g/ml) (min) (log.sub.10) (%) Normally aerated 25 20
5.1 77 Normally aerated + 25 20 5.3 94 1 mM GSH Deaerated 25 120
.gtoreq.6.1 91
Example 15
Improved Virus Kill in Platelets Treated with a Psoralen
Derivative: Effect of Quencher Addition Versus Deoxygenation
A standard blood bank platelet concentrate was treated with 25
.mu.g/ml of AMT and exposure to UVA (6.5 mW/CM.sup.2). Prior to
treatment, where indicated, oxygen in the normally aerated
platelets was exchanged for nitrogen by displacing the gas above
the solution using nitrogen, equilibrating for 1 minute, and
repeating the process three times. Alternately, 1 mM of the
quencher glutathione was added prior to treatment. The rate of
inactivation of vesicular stomatitis virus, added to the sample and
serving as a viral marker, was assessed throughout the duration of
UVA exposure. The aggregation response of the platelets was
assessed 24 hours following treatment in the presence of collagen.
The results indicate that virus kill occurred more quickly in the
normally aerated sample containing quencher than in the deaerated
sample (Table XII). Excellent platelet functional recovery was
achieved in each case when measured at 24 hours. Thus the use of
quencher avoids the tedious and potentially time consuming step of
gas exchange without sacrifice of virus kill or platelet
functionality.
TABLE XII VSV Titer (log.sub.10) Duration of UVA O.sub.2 +
(Minutes) 1 mM Glutathione Deaerated Start 5.0 5.0 10 3.1 -- 20 1.4
-- 30 .ltoreq.-0.5 -- 60 .ltoreq.-0.5 2.4 90 .ltoreq.-0.5 1.4 120
.ltoreq.-0.5 .ltoreq.-0.5
It will be appreciated that the instant specification is set forth
by way of illustration and not limitation, and that various
modifications and changes may be made without departing from the
spirit and scope of the present invention.
* * * * *